The biobased-economy aims to create a circular biotechnology ecosystem to transition from a fossil fuel-based to a sustainable industry based on biomass. For this, new microbial cell-factories are essential. We present the draft genome of the CEN.PK-derived Saccharomyces cerevisiae SpyCas9 expressing strain (IMX2600), that serve as chassis of new cell-factories.@en

An oxygen requirement for de novo biotin synthesis in Saccharomyces cerevisiae precludes the application of biotin-prototrophic strains in anoxic processes that use biotin-free media. To overcome this issue, this study explores introduction of the oxygen-independent Escherichia coli biotin-biosynthesis pathway in S. cerevisiae. Implementation of this pathway required expression of seven E. coli genes involved in fatty-acid synthesis and three E. coli genes essential for the formation of a pimelate thioester, key precursor of biotin synthesis. A yeast strain expressing these genes readily grew in biotin-free medium, irrespective of the presence of oxygen. However, the engineered strain exhibited specific growth rates 25% lower in biotin-free media than in biotin-supplemented media. Following adaptive laboratory evolution in anoxic cultures, evolved cell lines that no longer showed this growth difference in controlled bioreactors, were characterized by genome sequencing and proteome analyses. The evolved isolates exhibited a whole-genome duplication accompanied with an alteration in the relative gene dosages of biosynthetic pathway genes. These alterations resulted in a reduced abundance of the enzymes catalyzing the first three steps of the E. coli biotin pathway. The evolved pathway configuration was reverse engineered in the diploid industrial S. cerevisiae strain Ethanol Red. The resulting strain grew at nearly the same rate in biotin-supplemented and biotin-free media non-controlled batches performed in an anaerobic chamber. This study established an unique genetic engineering strategy to enable biotin-independent anoxic growth of S. cerevisiae and demonstrated its portability in industrial strain backgrounds.@en

Biotin, an important cofactor for carboxylases, is essential for all kingdoms of life. Since native biotin synthesis does not always suffice for fast growth and product formation, microbial cultivation in research and industry often requires supplementation of biotin. De novo biotin biosynthesis in yeasts is not fully understood, which hinders attempts to optimize the pathway in these industrially relevant microorganisms. Previous work based on laboratory evolution of Saccharomyces cerevisiae for biotin prototrophy identified Bio1, whose catalytic function remains unresolved, as a bottleneck in biotin synthesis. This study aimed at eliminating this bottleneck in the S. cerevisiae laboratory strain CEN.PK113-7D. A screening of 35 Saccharomycotina yeasts identified six species that grew fast without biotin supplementation. Overexpression of the S. cerevisiaeBIO1 (ScBIO1) ortholog isolated from one of these biotin prototrophs, Cyberlindnera fabianii, enabled fast growth of strain CEN.PK113-7D in biotin-free medium. Similar results were obtained by single overexpression of C. fabianii BIO1 (CfBIO1) in other laboratory and industrial S. cerevisiae strains. However, biotin prototrophy was restricted to aerobic conditions, probably reflecting the involvement of oxygen in the reaction catalyzed by the putative oxidoreductase CfBio1. In aerobic cultures on biotin-free medium, S. cerevisiae strains expressing CfBio1 showed a decreased susceptibility to contamination by biotin-auxotrophic S. cerevisiae This study illustrates how the vast Saccharomycotina genomic resources may be used to improve physiological characteristics of industrially relevant S. cerevisiaeIMPORTANCE The reported metabolic engineering strategy to enable optimal growth in the absence of biotin is of direct relevance for large-scale industrial applications of S. cerevisiae Important benefits of biotin prototrophy include cost reduction during the preparation of chemically defined industrial growth media as well as a lower susceptibility of biotin-prototrophic strains to contamination by auxotrophic microorganisms. The observed oxygen dependency of biotin synthesis by the engineered strains is relevant for further studies on the elucidation of fungal biotin biosynthesis pathways.@en

Chemically defined media for yeast cultivation (CDMY) were developed to support fast growth, experimental reproducibility, and quantitative analysis of growth rates and biomass yields. In addition to mineral salts and a carbon substrate, popular CDMYs contain seven to nine B-group vitamins, which are either enzyme cofactors or precursors for their synthesis. Despite the widespread use of CDMY in fundamental and applied yeast research, the relation of their design and composition to the actual vitamin requirements of yeasts has not been subjected to critical review since their first development in the 1940s. Vitamins are formally defined as essential organic molecules that cannot be synthesized by an organism. In yeast physiology, use of the term “vitamin” is primarily based on essentiality for humans, but the genome of the Saccharomyces cerevisiae reference strain S288C harbours most of the structural genes required for synthesis of the vitamins included in popular CDMY. Here, we review the biochemistry and genetics of the biosynthesis of these compounds by S. cerevisiae and, based on a comparative genomics analysis, assess the diversity within the Saccharomyces genus with respect to vitamin prototrophy.@en

Every century brings its own challenges, but the 21st century is the first in which a global transition towards circularity is required to ensure human existence on this planet. Exhaustion of planetary resources, such as oil and rare elements, must be prevented and sustainable circular value chains introduced into our industry and economy. In addition to new challenges, every century also brings new and unique solutions. Today, biotechnology may provide some of the most relevant solutions by providing scientists with the ability to decipher the code of life represented by an organism’s DNA as well as with the tools to edit this code. Especially fast-reproducing microorganisms have a great potential to serve as cell factories, which can convert renewable raw materials into chemicals, materials and food ingredients and thereby support a circular bio-based economy. Recently developed biotechnological tools enable us to rewrite (‘edit’) the blueprint for these microbial cell factories with unprecedented precisions and at unprecedented rates. A myriad of life forms evolved over billions of years to adapt to an incredibly diverse number of habitats on our planet, which led to an immense diversity in survival strategies and metabolic capabilities. Recombining these naturally occurring DNA codes and ‘novel-to-nature’ DNA sequences generated in laboratories offers unique possibilities for development of novel cell factories to address challenges in our century and beyond. Baker’s yeast, Saccharomyces cerevisiae is one of the most intensively studied microorganisms and, as a cell factory, has a long history of successful application in industrial applications. Its story of success began thousands of years ago when processes for production of wine, beer and bread-making were first invented and, over many centuries, improved. Application of yeasts probably started as serendipitous discovery rather than as an invention, when yeast cells from the environment ‘contaminated’ sugar-containing food products and, by accident, turned sugars into ethanol and carbon dioxide, thus yielding the first alcoholic beverages and rising dough. All essential nutrients that yeast require for growth and fermentation were either present in the food product or generated by other microorganisms that inadvertently entered these early fermentation processes. Such a co-existence of multiple microbial species is a natural phenomenon that helps organisms thrive, but in man-made industrial settings such undefined mixed populations are often difficult to control and optimize. When researchers discovered that pure cultures of individual yeast strains were very efficient in producing transport fuels and other interesting chemicals, they therefore developed growth media that contained all essential and non-essential nutrients required for optimal yeast growth, to make these yeast cell factories as productive as possible. For over a century now, yeast cell factories have been under continual development. Classical strain improvement strategies to obtain high-producing strains, later combined with recombinant-DNA technology (genetic engineering) brought microbial production systems to a next level and helped pave the way towards a sustainable bio-based industry. However, while studying and developing product pathways for yeast strains employed in these processes, the specific requirements of these hosts regarding essential nutrients (vitamins) did not always receive attention. Use of generic media, containing excess amounts of vitamins to ensure high productivity, increase overall production costs, complicate down-stream processing and increase contamination risks. The research described in this thesis explores genetic engineering strategies in which heterologous DNA sequences are introduced to improve vitamin synthesis under industrially relevant conditions, with the goal to enable development of fully vitamin-independent (prototrophic) S. cerevisiae strains. The research focusses on a number of compounds that are routinely added to synthetic media for cultivation of S. cerevisiae that, based on their role in human nutrition, are referred to as B-type vitamins. A special focus was laid upon one of the more expensive B vitamins, biotin. The pathway by which some S. cerevisiae strains synthesize biotin is still not completely resolved. By a combination of laboratory evolution, genome analysis and genetic engineering, different strategies were designed and tested to obtain biotin prototrophic and fully vitamin-independent S. cerevisiae strains…@en

Chemically defined media for cultivation of Saccharomyces cerevisiae strains are commonly supplemented with a mixture of multiple Class-B vitamins, whose omission leads to strongly reduced growth rates. Fast growth without vitamin supplementation is interesting for industrial applications, as it reduces costs and complexity of medium preparation and may decrease susceptibility to contamination by auxotrophic microbes. In this study, suboptimal growth rates of S. cerevisiae CEN.PK113-7D in the absence of pantothenic acid, para-aminobenzoic acid (pABA), pyridoxine, inositol and/or biotin were corrected by single or combined overexpression of ScFMS1, ScABZ1/ScABZ2, ScSNZ1/ScSNO1, ScINO1 and Cyberlindnera fabianii BIO1, respectively. Several strategies were explored to improve growth of S. cerevisiae CEN.PK113-7D in thiamine-free medium. Overexpression of ScTHI4 and/or ScTHI5 enabled thiamine-independent growth at 83% of the maximum specific growth rate of the reference strain in vitamin-supplemented medium. Combined overexpression of seven native S. cerevisiae genes and CfBIO1 enabled a maximum specific growth rate of 0.33 ± 0.01 h−1 in vitamin-free synthetic medium. This growth rate was only 17 % lower than that of a congenic reference strain in vitamin-supplemented medium. Physiological parameters of the engineered vitamin-independent strain in aerobic glucose-limited chemostat cultures (dilution rate 0.10 h−1) grown on vitamin-free synthetic medium were similar to those of similar cultures of the parental strain grown on vitamin-supplemented medium. Transcriptome analysis revealed only few differences in gene expression between these cultures, which primarily involved genes with roles in Class-B vitamin metabolism. These results pave the way for development of fast-growing vitamin-independent industrial strains of S. cerevisiae.@en